The use of various methods to detect the interactions between a cell and another entity (generally a drug or a drug candidate, a toxin, environmental contaminant, etc.) is well known in the art. A number of these cell based assay formats are widely available. These assays are used to detect the effect of a test compound on the expression of one or more marker molecules (generally proteins) of a cell.
Cell based assays generally measure a differentiative response requiring an immunoassay for a differentiation-specific marker molecule such as a protein on the cell wall or intracellular of the cell. Generally, at least a first ligand is added (first antibody or other entity) that binds to the marker molecule of the cell and either by itself or with a secondary ligand (such as a secondary antibody having a detectable marker) is capable of being detected, generally by fluorometry, colorometry or radioactivity.
Immunoassays are complicated by the need to remove unbound ligands before the detection step. Typically this has been accomplished with centrifugation wash protocols. The level of liquid after the addition of the one or more ligands is reduced and a wash buffer is added to the solution. This is then centrifuged and the supematant removed leaving the cells in a small volume of liquid. The wash steps are often repeated 3-8 times in order to ensure the substantial removal of the unbound materials.
This is a time consuming process and limited in the number of tubes which can processed at any given time by one person. It also is not easily if at all automation compatible. Additionally, the amount withdrawn and where it is drawn from varies and leads to loss of cells either through decantation or due to stress or death due to lack of liquid (if too much is taken). Likewise, the number of washes must be kept high to ensure sufficient background reagent is removed so an accurate measurement can be made.
US 2001/0055776 A1 suggested the use of vacuum with a multiwell filter plate to provide a higher throughput format. The low vacuum is difficult to control and each well varies in its response to the low vacuum. This leads to inconsistent results between wells with some wells running dry while others retain too much fluid leading to false positive signals. More importantly, cell recovery is substantially low, albeit in some cases may permit an assay using remaining low number of cells.
The lack of a high throughput, high cell viability/retention assay is a significant bottleneck in the realization of proteonomics' full potential for drug discovery. The present invention provides such a solution.